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Importance of MEK-1/-2 signaling in monocytic and granulocytic differentiation of myeloid cell lines

Leukemia volume 16, pages 683–692 (2002)Cite this article

Abstract

Activation of the MEK/ERK/MAP kinase signaling pathway promotes the proliferation and survival of hematopoietic cells. The kinases MEK-1, MEK-2, ERK-1/MAPK and ERK-2/MAPK are activated by phosphorylation at specific sites, and these events can be monitored using phospho-specific antibodies. In this report we examined the importance of the MEK/ERK/MAP kinase pathway in the monocytic and granulocytic differentiation of myeloid cell lines. Induction of monocytic differentiation in HL-60 cells by treatment with phorbol 12-myristate 13-acetate (PMA) led to rapid and sustained activation of MEK-1/-2, ERK-1/MAPK and ERK-2/MAPK, while induction of granulocytic differentiation by retinoic acid (RA) caused similar activation of MEK-1/-2 and ERK-2/MAPK, but not ERK-1/MAPK. The total levels of these kinases were not affected during the course of differentiation along either pathway. Pretreatment of cells with 5 μM of the MEK-1/-2-specific inhibitor U0126 abrogated PMA- or RA-induced activation of ERK-1/MAPK and ERK-2/MAPK. Importantly, pretreatment of HL-60 cells with U0126 was found to potently inhibit both monocytic and granulocytic differentiation, as assessed by cytochemical staining for non-specific esterase or nitroblue tetrazolium reduction, flow cytometric analysis of myeloid surface markers, and immunoblotting for the cell cycle inhibitor p21 WAF1/Cip1. Similar results were seen in U937 cells, where U0126 inhibited PMA-induced monocytic differentiation, and in 32D cells, where G-CSF-induced granulocytic differentiation was inhibited by U0126 pretreatment. Additional experiments revealed that inhibition of MEK-1/-2 in HL-60 cells resulted in nearly complete inhibition of differentiation-induced cell death during monocytic differentiation. By contrast, U0126 only partially inhibited cell death resulting from granulocytic differentiation. Taken together, our findings demonstrate that the MEK/ERK/MAP kinase signaling pathway is activated, and plays a critical role, during both monocytic and granulocytic differentiation of myeloid cell lines.

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Acknowledgements

We acknowledge the assistance of Bratislav and Jelena Janic with flow cytometric analyses. We appreciate the helpful discussions and troubleshooting tips given by Dr Vivian Liu. This work was supported by an American Cancer Society grant (RPG-99–203–01-LBC) to DEJ.

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  1. Department of Medicine, University of Pittsburgh and the University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

    MB Miranda & DE Johnson

  2. Department of Pharmacology, University of Pittsburgh and the University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

    TF McGuire & DE Johnson

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Miranda, M., McGuire, T. & Johnson, D. Importance of MEK-1/-2 signaling in monocytic and granulocytic differentiation of myeloid cell lines. Leukemia 16, 683–692 (2002). https://doi.org/10.1038/sj.leu.2402400

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